Maize event HCEM485, compositions and methods for detecting and use thereof

ABSTRACT

Maize event HCEM485 is provided, in which plants comprising the event are tolerant to exposure to a glyphosate herbicide. Maize genomic polynucleotides flanking the insert DNA providing glyphosate tolerance are provided. The plant or part thereof having the event comprises a junction region of the insert DNA and maize plant genomic sequences. Methods and primers and probes to detect the presence of the event are provided, as well as kits which employ such primers and probes.

REFERENCE TO RELATED APPLICATION

This application is a divisional of previously filed and co-pendingapplication U.S. Ser. No. 13/211,622, the contents of which areincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 3, 2011, isnamed 210010.txt and is 55,451 bytes in size.

BACKGROUND OF THE INVENTION

Glyphosate (N-phosphonomethylglycine) is a widely used active ingredientin herbicides. Glyphosate inhibits 5-enolpyruvyl-3-phosphoshikimic acidsynthase (EPSP synthase, or EPSPS). EPSPS is involved in the synthesisof aromatic amino acids in plant cells. Inhibition of EPSPS effectivelydisrupts protein synthesis and thereby kills the affected plant cells.Because glyphosate is non-selective, it kills both weeds and cropplants. Thus it is useful with crop plants when one can modify the cropplants to be resistant to glyphosate, allowing the desirable plants tosurvive exposure to the glyphosate.

Recombinant DNA technology has been used to isolate mutant EPSPsynthases that are glyphosate-resistant. Such glyphosate-resistantmutant EPSP synthase genes can be transformed into plants and conferglyphosate-resistance upon the transformed plants. By way of example, aglyphosate tolerant gene was isolated from Agrobacterium strain CP4 asdescribed in U.S. Pat. No. 5,633,435. The full length maize EPSPS geneis described at U.S. Pat. No. 7,045,684. It is imported to thechloroplast and the chloroplast transit peptide cleaved, producing themature EPSPS. See Herouet-Guicheney et al. (2009) “Safety evaluation ofthe double mutant 5-enolypyruvylshikimate-3-phosphate synthase (2mEPSPS)from maize that confers tolerance to glyphosate herbicide in transgenicplants” Regulatory Toxicology and Pharmacology, Vol. 54, Issue 2, pp143-153. Other glyphosate tolerant genes have been created through theintroduction of mutations. These include those isolated by Comai anddescribed at U.S. Pat. Nos. 5,094,945, 4,769,061 and 4,535,060. A singlemutant has been utilized, as described in 5,310,667 by substituting analanine residue for a glycine residue at between positions 80 and 120.Double mutants are also described at U.S. Pat. Nos. 6,225,114 and5,866,775 in which, in addition to the above mutation, a second mutation(a threonine residue for an alanine residue between positions 170 and210) is introduced into a wild-type EPSPS gene.

Other work resulted in the production of a glyphosate tolerant EPSPSmaize through the introduction of a double mutant EPSPS gene bearingmutations at residue 102 (changing threonine to isoleucine) and atresidue 106 (changing proline to serine) of the amino acid sequenceencoded by GenBank Accession No. X63374 and shown in U.S. Pat. Nos.6,566,587 and 6,040,497, each of which are incorporated herein byreference in their entirety.

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising et al.(1988) Ann. Rev. Genet. 22: 421-477, 1988). At the same time thepresence of the transgene at different locations in the genomeinfluences the overall phenotype of the plant in different ways. Forthis reason, it is often necessary to screen a large number of events inorder to identify an event characterized by optimal expression of anintroduced gene of interest. For example, it has been observed in plantsand in other organisms that there may be a wide variation in levels ofexpression of an introduced gene among events. There may also bedifferences in spatial or temporal patterns of expression, for example,differences in the relative expression of a transgene in various planttissues, that may not correspond to the patterns expected fromtranscriptional regulatory elements present in the introduced geneconstruct. It is also observed that the transgene insertion can affectthe endogenous gene expression. For these reasons, it is common toproduce hundreds to thousands of different events and screen thoseevents for a single event that has desired transgene expression levelsand patterns for commercial purposes. An event that has desired levelsor patterns of transgene expression is useful for introgressing thetransgene into other genetic backgrounds by sexual outcrossing usingconventional breeding methods. Progeny of such crosses maintain thetransgene expression characteristics of the original transformant. Thisstrategy is used to ensure reliable gene expression in a number ofvarieties that are well adapted to local growing conditions.

SUMMARY OF THE INVENTION

Compositions and methods related to cisgenic glyphosate tolerant maizeplants are provided. Specifically, the present invention provides maizeplants, plant parts, seeds and commodity products containing the HCEM485event which imparts tolerance to glyphosate. The maize plant harboringthe HCEM485 event at the recited chromosomal location comprisesgenomic/cisgene HCEM485 junction sequences having at least thepolynucleotide sequence of base pairs 367/368 of SEQ ID NO: 4, or whichin an embodiment comprises SEQ ID NO: 4, 12, 13, 14, 15, 16, or 17 orfragments thereof sufficient to identify the presence of the HCEM485event.

Methods and kits for detecting the presence of event HCEM485 areprovided, where the presence of a junction region is detected. In oneembodiment, a diagnostic amplicon is detected. The methods include useof one or more primers or a specific probe binding to a HCEM485 junctionregion. Also provided are representative seeds with the American Typeculture Collection (ATCC) with Accession No. PTA-12014 and plants, plantcells, plant parts, grain, food and feed and plant products and progenyderived therefrom. Progeny denotes the offspring of any generation of aparent plant comprising the HCEM485 Event. The characterization of thegenomic insertion site of event HCEM485 provides for an enhancedbreeding efficiency and enables the use of molecular markers to trackthe insert in the breeding populations and progeny thereof. Variousmethods and compositions for the identification, detection, and use ofthe maize event HCEM485 are provided. All references cited herein areincorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plasmid map of pHCEM used to produce the HCEM485 maizeline.

FIG. 2 shows a southern blot hybridization of HCEM with A/E and A/Cprobes.

FIG. 3 is a diagram of the process for producing plants for agronomicand phenotypic comparisons.

FIG. 4 shows the amplified nucleotide sequence of SEQ ID NO: 4 obtainedusing primer 302 (SEQ ID NO: 5). Primer 302 is underlined at thebeginning of the sequence, and its complementary region underlined atthe end of the sequence (SEQ ID NO: 6). The complementary region toPrimer 506 is shown in bold (SEQ ID NO: 11). The junction region atbases 367 and 368 is shown in bold and italics.

FIG. 5 is a diagram of the plasmid J4 showing genetic elements includingthe location of Primer 302 and its complementary sequence, the 3′ HCEMregion and flanking region of chromosome 10.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a novel transformation event of maize plantscomprising a transgene/cisgene providing tolerance to exposure toglyphosate herbicide. Compositions and methods related tocisgenicglyphosate-tolerant maize plants are provided. Specifically, thepresent invention provides maize plants having event HCEM485.

The HCEM event comprises a maize genomic DNA fragment comprising anEPSPS 5′ regulatory sequence, and a coding sequence encoding aglyphosate-tolerant EPSPS. The EPSPS 5′ regulatory sequence is operablylinked to the EPSPS coding sequence. The glyphosate-resistant EPSPSincludes a chloroplast transit peptide. The DNA fragment does notcontain a non-EPSPS enhancer.

The sequence providing tolerance to glyphosate exposure is described atU.S. Pat. No. 7,045,684 and Reissue No. RE41,943 incorporated herein byreference in its entirety. In U.S. Pat. No. 7,045,684, a genomic EPSPSfragment was isolated from maize as is described in Example 3,incorporated herein by reference in its entirety (SEQ ID NO: 1). The 6.0kb fragment includes an EPSPS 5′ regulatory sequence (the sequencebefore nucleotide 1868), an EPSPS coding sequence (from nucleotide 1868to nucleotide 5146), and an EPSPS 3′ regulatory sequence (the sequenceafter nucleotide 5146). The EPSPS coding sequence also encodes achloroplast transit peptide (from nucleotide 1868 to nucleotide 2041).The sequence encoding this chloroplast transit peptide can be predictedusing the computer program PSORT maintained on the public accessibleGenomeNet at Kyoto University, Japan. Subsequently two mutations wereintroduced into the corn wild-type EPSPS gene; the first a cytosine tothymine substitution at nucleotide 2886, and the second a cytosine tothymine substitution at nucleotide 2897 (SEQ ID NO: 2). The mutated gene(referred to as HCEM) encodes mutant protein which is SEQ ID NO: 3 withthe residue at position 164 changed from threonine in the wild-type toisoleucine (Thr to Ile) and at position 168 changed from proline toserine (Pro to Ser). The resulting mutated amino acid sequence wasglyphosate resistant.

The mutated nucleotide sequence of SEQ ID NO: 2 includes 2mEPSPS, thatis, in this instance, the native corn EPSPS promoter, coding regions orexons (containing the two mutations), introns and 3′ terminator region.This was introduced into a corn plant as described in Example 4 of the'684 patent, incorporated herein by reference in its entirety, andresulted in event HCEM485 located on chromosome 10. Resistance toglyphosate in regenerants was confirmed by spraying them with glyphosateat commercial rates. Seed from the regenerants segregated 3:1 forresistance as would be expected with Mendelian inheritance of atransgene. Seeds from backcrossed individuals segregated 1:1.

FIG. 4 shows the 1112 base pair amplified nucleotide sequence (SEQ IDNO: 4, referred to also as J4) obtained using the flanking region of the2mEPSPS insert. The amplified region includes as base pairs 1-25 primer302 (SEQ ID NO: 5) which is a portion of the 3′ fragment of the insertedDNA. The 302 primer was able to amplify the 3′ region of the insert. Thefinal 25 base pairs of J4 (underlined in FIG. 4 and which is SEQ ID NO:6) is the complement to the 302 primer. The first 367 base pairs (SEQ IDNO: 7) of the J4 amplified sequence have 100% homology to the 3′sequence of the HCEM fragment and thus represent this region of theinsert. The remaining portion of the sequence, base pairs 368-1112,represent chromosome 10 of the maize genome (SEQ ID NO: 8) with by368-1092 having 100% homology to chromosome 10 (SEQ ID NO: 9). Thus thejunction between the insert and maize genome is base pair 367-368.Primer 506 (SEQ ID NO: 10) can also be used with primer 302, asdescribed below to amplify a fragment. The region complementary toPrimer 506 is shown in bold face in FIG. 4 and is SEQ ID NO: 11. Themaize plant harboring the HCEM485 event at the recited chromosomallocation comprises genomic/inserted DNA junctions having at least thepolynucleotide sequence of the junction 367/368. The maize plantharboring the HCEM485 event at the recited chromosomal locationcomprises genomic/cisgenic HCEM485 junction sequences having at leastthe polynucleotide sequence of base pairs 367/368 of SEQ ID NO: 4, orwhich in an embodiment comprises SEQ ID NO: 4, 12, 13, 14 15, 16, or 17or fragments thereof sufficient to identify the presence of the HCEM485event. In one embodiment of the invention at least one primer describedhere produces a diagnostic amplicon that is an amplicon that detects thepresence of the HCEM485 event. The amplicon will encompass an amplifiedproduct comprising at least one HCEM junction sequence.

The characterization of the genomic insertion site of the HCEM485 eventprovides for an enhanced breeding efficiency and enables the use ofmolecular markers to track the insert in the breeding populations andprogeny thereof. Various methods and compositions for theidentification, detection, and use of the maize HCEM485 event areprovided herein. As used herein, the term “event HCEM485 specific”refers to a polynucleotide sequence which is suitable fordiscriminatively identifying event HCEM485 in plants, plant cells, plantparts, grain, food and feed and plant products and progeny derivedtherefrom, or in plant materials and plant products such as, but notlimited to, food or feed products (fresh or processed) comprising, orderived from plant material. The invention further encompasses acommodity product produced from seed comprising even HCEM485. Suchcommodity product includes grain, meal, flour, flakes, oil, food or feedproducts and the like.

Compositions further include seed deposited as Patent Deposit Nos.PTA-12014 and plants, plant cells, and seed derived therefrom.Applicant(s) have made a deposit of at least 2500 seeds of maize eventHCEM485 with the American Type Culture Collection (ATCC), Manassas, Va.20110-2209 USA, on Jul. 29, 2011 and the deposits were assigned ATCCDeposit No. PTA-12014. These deposits will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. These depositswere made merely as a convenience for those of skill in the art and arenot an admission that a deposit is required under 35 U.S.C.§112. Theseeds deposited with the ATCC on Jul. 29, 2011 were taken from thedeposit maintained by Stine Seed, Inc., 22555 Laredo Trail Adel, Iowa50003. Access to this deposit will be available during the pendency ofthe application to the Commissioner of Patents and Trademarks andpersons determined by the Commissioner to be entitled thereto uponrequest. Upon allowance of any claims in the application, theApplicant(s) will make available to the public, pursuant to 37 C.F.R.§1.808, sample(s) of the deposit of at least 2500 seeds of hybrid maizeB485 with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209. This deposit of seed of maize eventHCEM485 will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicant(s) have satisfied all the requirements of 37C.F.R. §§1.801-1.809, including providing an indication of the viabilityof the sample upon deposit. Applicant(s) have no authority to waive anyrestrictions imposed by law on the transfer of biological material orits transportation in commerce. Applicant(s) do not waive anyinfringement of their rights granted under this patent or rightsapplicable to event HCEM485 under the Plant Variety Protection Act (7USC §2321 et seq.). Unauthorized seed multiplication prohibited. Theseed may be regulated.

As used herein, the term “maize” means any maize plant and includes allplant varieties that can be bred with maize. As used herein, the termplant includes plant cells, plant organs, plant protoplasts, plant celltissue cultures from which plants can be regenerated, plant calli, plantclumps, and plant cells that are intact in plants or parts of plantssuch as embryos, pollen, ovules, seeds, leaves, flowers, branches,fruit, stalks, roots, root tips, anthers, and the like. Grain isintended to mean the mature seed produced by commercial growers forpurposes other than growing or reproducing the species. Progeny,variants, and mutants of the regenerated plants are also included withinthe scope of the invention, provided that these parts comprise a HCEM485event.

A transgenic/cisgenic “event” is produced by transformation of plantcells with a heterologous DNA construct(s), including a nucleic acidexpression cassette that comprises a transgene of interest, theregeneration of a population of plants resulting from the insertion ofthe transgene into the genome of the plant, and selection of aparticular plant characterized by insertion into a particular genomelocation. An event is characterized phenotypically by the expression ofthe transgene(s). When referring here to a transgene of interest ismeant to encompass the cisgene of interest, that is, a gene in which thenucleic acid molecules introduced into the plant are sequences found ina wild-type plant, but which have been introduced into the plant byhuman intervention. The term “event” also refers to progeny produced bya sexual outcross between the transformant and another variety thatinclude the heterologous DNA. Even after repeated back-crossing to arecurrent parent, the inserted DNA and flanking DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant comprising the inserted DNA and flanking sequenceimmediately adjacent to the inserted DNA that would be expected to betransferred to a progeny that receives inserted DNA including thetransgene of interest as the result of a sexual cross of one parentalline that includes the inserted DNA (e.g., the original transformant andprogeny resulting from selfing) and a parental line that does notcontain the inserted DNA.

An elite event is one in which the presence of the heterologous DNA doesnot adversely impact agronomic and other desired characteristics of theplant and which is stably inherited. A plant and plant material maycomprise one or more events in its genome.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can comprise either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process, e.g. fragments associated with thetransformation event. A “flanking region” or “flanking sequence” as usedherein refers to a sequence of at least 20, 30, 50, 100, 200, 300, 400,1000, 1500, 2000, 2500, or 5000 base pair or greater or any amountin-between which is located either immediately upstream of andcontiguous with or immediately downstream of and contiguous with theoriginal insert DNA molecule. Non-limiting examples of the flankingregions of the HCEM485 event comprise polynucleotide sequences that areset forth in SEQ ID NO: 8 and 9 and variants and fragments thereof.

During the process of introducing an insert into the genome of plantcells, it is not uncommon for some deletions or other alterations of theinsert and/or genomic flanking sequences to occur. Thus, the relevantsegment of the DNA sequence provided herein might comprise some minorvariations. The same is true for the flanking sequences provided herein.Thus, a plant comprising a polynucleotide having some range of identitywith the subject flanking and/or insert sequences is within the scope ofthe subject invention. Identity to the sequence of the present inventioncan be a polynucleotide sequence having at least 65% sequence identity,more preferably at least 70% sequence identity, more preferably at least75% sequence identity, more preferably at least 80% identity, and morepreferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified ordescribed herein. Hybridization and hybridization conditions as providedherein can also be used to define such plants and polynucleotidesequences of the subject invention. The sequence which comprises theflanking sequences plus the full insert sequence can be confirmed withreference to the deposited seed.

Transformation procedures leading to random integration of the foreignDNA will result in transformants containing different flanking regionscharacteristic of and unique for each transformant. A “junction” is apoint where two specific DNA fragments join. For example, a junctionexists where insert DNA joins flanking DNA. A junction point also existsin a transformed organism where two DNA fragments join together in amanner that is modified from that found in the native organism. As usedherein, “junction DNA” refers to DNA that comprises a junction point.Non-limiting examples of junction DNA from the HCEM485 event set areforth in SEQ ID NO: 4, 12, 13, 14, 15, 16 and 17 and variants andfragments thereof. The amplicon produced using these primers in the DNAamplification method is diagnostic for maize event HCEM485.

In an embodiment, a HCEM485 plant can be bred by first sexually crossinga first parental maize plant grown from the HCEM485 maize plant (orprogeny thereof derived from transformation with the expressioncassettes of the embodiments of the present invention that conferherbicide tolerance) and a second parental maize plant that lacks theherbicide tolerance phenotype, thereby producing a plurality of firstprogeny plants; and then selecting at least one first progeny plant thatdisplays the desired herbicide tolerance; and selfing the first progenyplant, thereby producing a plurality of second progeny plants; and thenselecting from the second progeny plants which display the desiredherbicide tolerance. These steps can further include the back-crossingof the first herbicide tolerant progeny plant or the second herbicidetolerant progeny plant to the second parental maize plant or a thirdparental maize plant, thereby producing a maize plant that displays thedesired herbicide tolerance. It is further recognized that assayingprogeny for phenotype is not required. Various methods and compositions,as disclosed elsewhere herein, can be used to detect and/or identify theHCEM485 event.

Two different transgenic plants can also be sexually crossed to produceoffspring that contain two independently segregating added, exogenousgenes. Selfing of appropriate progeny can produce plants that arehomozygous for both added, exogenous genes. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in one of several references, e.g., Poehlman (1995)Breeding Field Crops. AVI Publication Co., Westport Conn., 4^(th) Edit.and Fehr (1987), in Breeding Methods for Cultivar Development, Wilcos J.ed., American Society of Agronomy, Madison Wis.

The term “germplasm” refers to an individual, a group of individuals, ora clone representing a genotype, variety, species or culture, or thegenetic material thereof. A “line” or “strain” is a group of individualsfrom a common ancestry. Inbred lines are the product of inbreeding,typically five or more generations of self-pollinations and selectionand is a true breeding strain. A “variety” is a subdivision of aspecies, a group of similar plants that by structural and/or agronomicfeatures can be identified from other varieties within the same species.

Inbred maize lines are typically developed for use in the production ofmaize hybrids and for use as germplasm in breeding populations for thecreation of new and distinct inbred maize lines. Inbred maize lines areoften used as targets for the introgression of novel traits throughtraditional breeding and/or molecular introgression techniques. Inbredmaize lines need to be highly homogeneous, homozygous and reproducibleto be useful as parents of commercial hybrids. Many analytical methodsare available to determine the homozygosity and phenotypic stability ofinbred lines. The phrase “hybrid plants” refers to plants which resultfrom a cross between genetically different individuals. The term“crossed” or “cross” in the context of this invention means the fusionof gametes, e.g., via pollination to produce progeny (i.e., cells,seeds, or plants) in the case of plants. The term encompasses bothsexual crosses (the pollination of one plant by another) and selfing(self-pollination, i.e., when the pollen and, ovule are from the sameplant). The term “introgression” refers to the transmission of a desiredallele of a genetic locus from one genetic background to another. In onemethod, the desired alleles can be introgressed through a sexual crossbetween two parents, wherein at least one of the parents has the desiredallele in its genome.

In some embodiments, the event can be “stacked” with other traits,including, for example, agronomic traits and/or insect-inhibitoryproteins and/or resistance to the same or other herbicides. Stackingrefers to combining traits into a line. One method is to transform aplant with two or more genes at the same time, or sequentially. Afurther method is to cross parents having the trait of interest andselecting progeny with the combined traits. Two or more different traitsmay be combined with such a process. In some embodiments, thepolynucleotide conferring the maize HCEM485 event of the invention areengineered into a molecular stack.

In an embodiment, the maize HCEM485 event of the invention comprise oneor more traits of interest, and in more specific embodiments, the plantis stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired combination of traits. A trait,as used herein, refers to the phenotype derived from a particularsequence or groups of sequences. Examples, without intending to belimiting, of other traits with which the event can be combined includeother herbicide tolerance providing genes, such as combination withother genes encoding glyphosate tolerance, (such as glyphosate oxidase(GOX) or glyphosate acetyl transferase (GAT)); glufosinate tolerance (asthrough use of the bar or pat gene); acetolactate synthase inhibitingtolerance as with imidazolinones, sulfonylureas, triazolopyrimidinesulfonanilide; bromozynil tolerance; tolerance to inhibitors of HPPD(4-hydroxylphenyl-pyruvate-dioxygenase) enzyme; tolerance to herbicidesconverted to phenoxyaceate auxin (such as 2,4-D) as with the aad-12 geneand the like. Still further examples include stacking polynucleotidesencoding polypeptides having pesticidal and/or insecticidal activity,such as Bacillus thuringiensis toxic proteins such as the Cry proteinencoding nucleotide genes (such as Cry1A, Cry1A, Cry1F, Cry1C, forexample) or vegetative insecticidal proteins (such as VIP3 encodinggenes); genes providing stress tolerance, fungal tolerance, or otherdesirable traits such as increased yield, particular oil profiles andany of a variety of desirable traits. It will be understood by oneskilled in the art that such traits for combination can be the result ofa molecular stack by combining with other transgenes, or combining withother nontransgenic traits.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology, orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes or contained on the sametransformation cassette. Expression of the sequences can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system. Amultitude of methods for site specific recombination are available toone skilled in the art, including, by way of example without limitation,introducing FRT sites in the FLP/FRT system and/or Lox sites that may beused in the Cre/Lox system. For example, see Lyznik, et al., (2003)“Site-Specific Recombination for Genetic Engineering in Plants,” PlantCell Rep 21:925-932.

Deoxyribonucleic acid (DNA) is a polymer comprising four mononucleotideunits, (D)dAMP (2′-(D) deoxyadenosine-5-monophosphate), dGMP(2′-(D)deoxyguanosine-5-monophosphate), dCMP(2′-(D)deoxycytosine-5-monophosphate) and dTMP(2′-(D)deoxycytosine-5-monophosphate) linked in various sequences by3′,5′-phosphodiester bridges. The structural DNA consists of multiplenucleotide triplets called “codons” which code for the amino acids. Thecodons correspond to the various amino acids as follows: Arg (CGA, CGC,CGG, CGT, AGA, AGG); Leu (CTA, CTC, CTG, CTT, TTA, TTG); Ser (TCA, TCC,TCG, TCT, AGC, AGT); Thr (ACA, ACC, ACG, ACT); Pro (CCA, CCC, CCG, CCT);Ala (GCA, GCC, GCG, GCT); Gly (GGA, GGC, GGG, GGT); Ile (ATA, ATC, ATT);Val (GTA, GTC, GTG, GTT); Lys (AAA, AAG); Asn (AAC, AAT); Gln (GAA,CAG); His (CAC, CAT); Glu (GAA, GAG); Asp (GAC, GAT); Tyr (TAC, TAT);Cys (TGC, TGT); Phe (TTC, TTT); Met (ATG); and Trp (UGG). Moreover, dueto the redundancy of the genetic code (i.e., more than one codon for allbut two amino acids), there are many possible DNA sequences which maycode for a particular amino acid sequence.

As used herein, the use of the term “polynucleotide” is not intended tolimit the present invention to polynucleotides comprising DNA. Those ofordinary skill in the art will recognize that polynucleotides, cancomprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like. Unlessotherwise indicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g. degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081;Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Rossolini et al.(1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

A HCEM485 plant comprises an expression cassette having a sequenceencoding a mutant EPSPS that provides tolerance to exposure toglyphosate, the EPSPS 5′ regulatory sequence and chloroplast transitpeptide.

The term introduced in the context of inserting a nucleic acid into acell, includes transfection or transformation or transduction andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). When referring tointroduction of a nucleotide sequence into a plant is meant to includetransformation into the cell, as well as crossing a plant having thesequence with another plant, so that the second plant contains theheterologous sequence, as in conventional plant breeding techniques.Such breeding techniques are well known to one skilled in the art. For adiscussion of plant breeding techniques, see Poehlman (1995) supra.Backcrossing methods may be used to introduce a gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPoehlman, supra, and Plant Breeding Methodology, edit. Neal Jensen, JohnWiley & Sons, Inc. (1988). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a plantis obtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

In specific embodiments, the polynucleotides of the invention comprisethe junction DNA sequence set forth in SEQ ID NO: 4, 12, 13, 14, 15, 16,or 17 or variants and fragments thereof. In specific embodiments,methods of detection described herein amplify a polynucleotidecomprising the junction of the HCEM485 specific event. Fragments andvariants of junction DNA sequences are suitable for discriminativelyidentifying event HCEM485. As discussed elsewhere herein, such sequencesfind use as primers and/or probes.

In other embodiments, the polynucleotides of the invention comprisepolynucleotides that can detect a HCEM485 event or a HCEM485 specificregion. Such sequences include any polynucleotide set forth in SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, or 22 or variants and fragments thereof. Fragments and variantsof polynucleotides that detect a HCEM485 event or a HCEM485 specificregion are suitable for discriminatively identifying event HCEM485. Asdiscussed elsewhere herein, such sequences find use as primers and/orprobes. In one embodiment further provided are isolated DNA nucleotideprimer sequences or kits comprising or consisting of a sequence setforth in SEQ ID NO: 5, 6, 10, 11, 18, 19, or 20 or a complement thereof.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide.

It is to be understood that as used herein the term“transgenic/cisgenic” includes any cell, cell line, callus, tissue,plant part, or plant, the genotype of which has been altered by thepresence of a heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic/cisgenic.

Various methods and compositions for identifying event HCEM485 areprovided. Such methods find use in identifying and/or detecting aHCEM485 event in any biological material. Such methods include, forexample, methods to confirm seed purity and methods for screening seedsin a seed lot for a HCEM485 event. In one embodiment, a method foridentifying event HCEM485 in a biological sample is provided andcomprises contacting the sample with a primer or a first and a secondprimer; and, amplifying a polynucleotide comprising a HCEM485 specificregion (a region within the flanking region of the event and preferablyalso comprising part of the insert DNA contiguous therewith).

A biological sample can comprise any sample in which one desires todetermine if DNA having event HCEM485 is present. For example, abiological sample can comprise any plant material or material comprisingor derived from a plant material such as, but not limited to, food orfeed products. As used herein, “plant material” refers to material whichis obtained or derived from a plant or plant part. In specificembodiments, the biological sample comprises a maize tissue.

The nucleotide sequences of the invention can also be used as molecularmarkers such as RFLP, AFLP, RAPD markers, SNPs and SSRs to identify theherbicide resistance trait where a plant is progeny of a parent havingthe HCEM485 Event.

Thus, in one specific embodiment, a method of detecting the presence ofmaize event HCEM485 or progeny thereof in a biological sample isprovided. The method comprises (a) extracting a DNA sample from thebiological sample; (b) providing a primer or pair of DNA primermolecules, including, but not limited to, any combination of sequencesin SEQ ID NO: 5, 6, 10, 11, 18 and 19 or a complement thereof (c)providing DNA amplification reaction conditions; (d) performing the DNAamplification reaction, thereby producing a DNA amplicon molecule; and(e) detecting the DNA amplicon molecule, wherein the detection of saidDNA amplicon molecule in the DNA amplification reaction indicates thepresence of maize event HCEM485. In order for a nucleic acid molecule toserve as a primer or probe it needs only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

Further provided are methods of detecting the presence of DNAcorresponding to the HCEM485 event in a sample. In one embodiment, themethod comprises (a) contacting the biological sample with apolynucleotide probe that hybridizes under stringent hybridizationconditions with DNA from maize event HCEM485 and specifically detectsthe HCEM485 event; (b) subjecting the sample and probe to stringenthybridization conditions; and (c) detecting hybridization of the probeto the DNA, wherein detection of hybridization indicates the presence ofthe HCEM485 event.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed, vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 1989; Current Protocols in Molecular Biology, ed. Ausubelet al., Greene Publishing and Wiley-Interscience, New York, 1992 (withperiodic updates); and Innis et al., PCR Protocols: A Guide to Methodsand Applications, Academic Press: San Diego, 1990. PCR primer pairs canbe derived from a known sequence, for example, by using computerprograms intended for that purpose such as the PCR primer analysis toolin Vector NTI version 6 (Informax Inc., Bethesda Md.); PrimerSelect(DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). Additionally, thesequence can be visually scanned and primers manually identified usingguidelines known to one of skill in the art.

As used herein, a “probe” is an isolated polynucleotide to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such aprobe is complementary to a strand of a target polynucleotide, in thecase of the present invention, to a strand of isolated DNA from maizeevent HCEM485 whether from a maize plant or from a sample that includesDNA from the event. Probes according to the present invention includenot only deoxyribonucleic or ribonucleic acids but also polyamides andother probe materials that can specifically detect the presence of thetarget DNA sequence.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primers and primer pairs of the invention refer to their usefor amplification of a target polynucleotide, e.g., by the polymerasechain reaction (PCR) or other conventional nucleic-acid amplificationmethods. “PCR” or “polymerase chain reaction” is a technique used forthe amplification of specific DNA segments (see, U.S. Pat. Nos.4,683,195 and 4,800,159; herein incorporated by reference). Anycombination of primers (i.e., SEQ ID NO: 5, 6 10, 11, 18 or 19)disclosed herein can be used such that the pair allows for the detectionof a HCEM485 event or specific region. Primer 302 (SEQ ID NO: 5) may beused alone, and in a preferred embodiment, is used with low stringentconditions. Non-limiting examples of primer pairs include SEQ ID NOS: 5and 10 and SEQ ID NOS: 18 and 19.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide having a HCEM485 event. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Generally, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500polynucleotides or more in length. Such probes and primers can hybridizespecifically to a target sequence under high stringency hybridizationconditions. Probes and primers according to embodiments of the presentinvention may have complete DNA sequence identity of contiguousnucleotides with the target sequence, although probes differing from thetarget DNA sequence and that retain the ability to specifically detectand/or identify a target DNA sequence may be designed by conventionalmethods. Accordingly, probes and primers can share about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity or complementarity to thetarget polynucleotide (i.e., SEQ ID NO: 1-20), or can differ from thetarget sequence by 1, 2, 3, 4, 5, 6 or more nucleotides. Probes can beused as primers, but are generally designed to bind to the target DNA orRNA and are not used in an amplification process. In one non-limitingembodiment, a probe can comprises a polynucleotide encoding the HCEMsequence or any variant or fragment of these sequences.

Any primer can be employed in the methods of the invention that allows aHCEM485 specific region to be amplified and/or detected. In anembodiment the primer comprises the sequence of or a fragment of apolynucleotide of SEQ ID NO: 2, 4, or 7 and shares sufficient sequenceidentity or complementarity to the polynucleotide to amplify the HCEM485specific region. For example, Primer 302, SEQ ID NO: 5 may be used aloneunder low stringent conditions. In another embodiment a primer pair canbe used which can comprise the sequence of or a fragment of SEQ ID NO:2, 5 or 7 and the sequence of or a fragment or variant of SEQ ID NO: 8or 9.

In still further embodiments, the first and the second primer cancomprise any one or any combination of the sequences set forth in SEQ IDNO: 5, 6, 10, 11, 18 or 19. The primers can be of any length sufficientto amplify a HCEM485 specific region including, for example, at least 6,7, 8, 9, 10, 15, 20, 15, or 30 or about 7-10, 10-15, 15-20, 20-25,25-30, 30-35, 35-40, 40-45 nucleotides or longer.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” or can itselfbe detected for identifying event HCEM485 in biological samples.Alternatively, a probe of the invention can be used during the PCRreaction to allow for the detection of the amplification event (i.e., aTaqMan® probe or an MGB probe, so called real-time PCR). When the probeis hybridized with the polynucleotides of a biological sample underconditions which allow for the binding of the probe to the sample, thisbinding can be detected and thus allow for an indication of the presenceof event HCEM485 in the biological sample. Such identification of abound probe has been described in the art. In an embodiment of theinvention, the specific probe is a sequence which, under optimizedconditions, hybridizes specifically to a region within the 5′ or 3′flanking region of the event and also comprises a part of the foreignDNA contiguous therewith. The specific probe may comprise a sequence ofat least 80%, between 80 and 85%, between 85 and 90%, between 90 and95%, and between 95 and 100% identical (or complementary) to a specificregion of the HCEM485 event.

Any of the polynucleotides and fragments and variants thereof employedin the methods and compositions of the invention can share sequenceidentity to a region of the transgene insert of the HCEM485 event, ajunction sequence of the HCEM485 event or a flanking sequence of theHCEM485 event. Methods to determine the relationship of varioussequences are known. As used herein, “reference sequence” is a definedsequence used as a basis for sequence comparison. A reference sequencemay be a subset or the entirety of a specified sequence; for example, asa segment of a full-length cDNA or gene sequence, or the complete cDNAor gene sequence. As used herein, “comparison window” makes reference toa contiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two polynucleotides. Generally, the comparison window is at least20 contiguous nucleotides in length, and optionally can be 30, 40, 50,100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Optimalalignment of sequences for comparison can use any means to analyzesequence identity (homology) known in the art, e.g., by the progressivealignment method of termed “PILEUP” (Morrison, Mol. Biol. Evol.14:428-441 (1997), as an example of the use of PILEUP); by the localhomology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482 (1981));by the homology alignment algorithm of Needleman & Wunsch (J. Mol. Biol.48:443 (1970)); by the search for similarity method of Pearson (Proc.Natl. Acad. Sci. USA 85: 2444 (1988)); by computerized implementationsof these algorithms (e.g., GAP, BEST FIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Gene program byIntelligenetics, Mountain View, Calif., described by, e.g., Higgins,Gene 73: 237-244 (1988); Corpet, Nucleic Acids Res. 16:10881-10890(1988); Huang, Computer Applications in the Biosciences 8:155-165(1992); and Pearson, Methods in Mol. Biol. 24:307-331 (1994); Pfam(Sonnhammer, Nucleic Acids Res. 26:322-325 (1998); TreeAlign (Hein,Methods Mol. Biol. 25:349-364 (1994); MEG-ALIGN, and SAM sequencealignment computer programs; or, by manual visual inspection.

Another example of algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul et al,J. Mol. Biol. 215: 403-410 (1990). The BLAST programs (Basic LocalAlignment Search Tool) of Altschul, S. F., et al., (1993) J. Mol. Biol.215:403-410) searches under default parameters for identity to sequencescontained in the BLAST “GENEMBL” database. A sequence can be analyzedfor identity to all publicly available DNA sequences contained in theGENEMBL database using the BLASTN algorithm under the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational. Center for Biotechnology Information, world wide webncbi.nlm.nih.gov/; see also Zhang, Genome Res. 7:649-656 (1997) for the“PowerBLAST” variation. This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence that either match or satisfy some positive valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, J. Mol. Biol. 215: 403-410 (1990)). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919 (1992)) alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. The term BLAST refers tothe BLAST algorithm which performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin, Proc. Natl. Acad.Sci. USA 90:5873-5787 (1993). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

In an embodiment, GAP (Global Alignment Program) can be used. GAP usesthe algorithm of Needleman and Wunsch J. Mol. Biol. 48:443-453 (1970) tofind the alignment of two complete sequences that maximizes the numberof matches and minimizes the number of gaps. Default gap creationpenalty values and gap extension penalty values in the commonly usedVersion 10 of the Wisconsin Package® (Accelrys, Inc., San Diego, Calif.)for protein sequences are 8 and 2, respectively. For nucleotidesequences the default gap creation penalty is 50 while the default gapextension penalty is 3. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. A generalpurpose scoring system is the BLOSUM62 matrix (Henikoff and Henikoff,Proteins, 17: 49-61 (1993)), which is currently the default choice forBLAST programs. BLOSUM62 uses a combination of three matrices to coverall contingencies. Altschul, J. Mol. Biol. 36: 290-300 (1993), hereinincorporated by reference in its entirety and is the scoring matrix usedin Version 10 of the Wisconsin Package® (Accelrys, Inc., San Diego,Calif.) (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915).

As used herein, “sequence identity” or “identity” in the context of twonucleic acid sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleotide amplification of a target polynucleotide that is part ofa nucleic acid template. For example, to determine whether a maize plantresulting from a sexual cross contains the HCEM485 event, DNA extractedfrom the maize plant tissue sample may be subjected to a polynucleotideamplification method using a primer diagnostic for a HCEM485 or a primerpair that includes a first primer derived from flanking sequenceadjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the HCEM485 event DNA.In specific embodiments, the amplicon comprises a HCEM485 junctionpolynucleotide (i.e., SEQ ID NO: 4, 12, 13, 14, 15, 16, or 17). By“diagnostic” for a HCEM485 event the use of any method or assay whichdiscriminates between the presence or the absence of a HCEM485 event ina biological sample is intended. Alternatively, the second primer may bederived from the flanking sequence. In still other embodiments, primerpairs can be derived from flanking sequence on both sides of theinserted DNA so as to produce an amplicon that includes the entireinsert polynucleotide of the expression construct as well as thesequence flanking the transgenic insert. The amplicon is of a length andhas a sequence that is also diagnostic for the event (i.e., has ajunction DNA from a HCEM485 event). The amplicon may range in lengthfrom the combined length of the primer pairs plus one nucleotide basepair to any length of amplicon producible by a DNA amplificationprotocol. A member of a primer pair derived from the flanking sequencemay be located a distance from the inserted DNA sequence, this distancecan range from one nucleotide base pair up to the limits of theamplification reaction, or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm the disclosed sequences byconventional methods, e.g., by re-cloning and sequencing such sequences.The polynucleotide probes and primers of the present inventionspecifically detect a target DNA sequence. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of DNA from a transgenic event in a sample. By “specificallydetect” it is intended that the polynucleotide can be used either as aprimer to amplify a HCEM485 specific region or the polynucleotide can beused as a probe that hybridizes under stringent conditions to apolynucleotide from a HCEM485 event. The level or degree ofhybridization which allows for the specific detection of a HCEM485 eventor a specific region of a HCEM485 event is sufficient to distinguish thepolynucleotide with the HCEM485 specific region from a polynucleotidelacking this region and thereby allow for discriminately identifying aHCEM485 event. By “sharing sufficient sequence identity orcomplementarity to allow for the amplification of a HCEM485 specificevent” is intended the sequence shares at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity or complementarity to a fragment or across the fulllength of the polynucleotide from the HCEM485 specific region.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which one primer having the corresponding wild-typesequence (or its complement) and another primer having the correspondingHCEM485 inserted DNA sequence would bind and preferably to produce anidentifiable amplification product (the amplicon) having a HCEM485specific region in a DNA thermal amplification reaction. In a PCRapproach, oligonucleotide primers can be designed for use in PCRreactions to amplify a HCEM485 specific region. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Methods ofamplification are further described in U.S. Pat. Nos. 4,683,195,4,683,202 and Chen et al. (1994) PNAS 91:5695-5699. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the embodiments of the present invention. It isunderstood that a number of parameters in a specific PCR protocol mayneed to be adjusted to specific laboratory conditions and may beslightly modified and yet allow for the collection of similar results.These adjustments will be apparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allowsfor the detection of the HCEM485 event or a HCEM485 specific region. Forexample, the amplicon can be about 10, 50, 100, 200, 300, 500, 700, 100,2000, 3000, 4000, 5000 nucleotides in length or longer. In specificembodiments, the specific region of the HCEM485 event is detected.

As discussed elsewhere herein, any method to amplify the HCEM485 eventor specific region can be employed, including for example, PolymeraseChain Reaction (PCR) or real time PCR (RT-PCR). See, for example, Livaket al. (1995a) “Oligonucleotides with fluorescent dyes at opposite endsprovide a quenched probe system for detecting PCR product and nucleicacid hybridization” PCR methods and Application. 4:357-362; U.S. Pat.No. 5,538,848; U.S. Pat. No. 5,723,591; Applied Biosystems User BulletinNo. 2, “Relative Quantitation of Gene Expression,” P/N 4303859; and,Applied Biosystems User Bulletin No. 5, “Multiplex PCR with TaqMan® VICprobes,” P/N 4306236.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide having a HCEM485specific event is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probeconditions under which a probe will hybridize to its target sequence toa detectably greater degree than to other sequences (e.g., at least2-fold over background) are intended. Regarding the amplification of atarget polynucleotide (e.g., by PCR) using a particular amplificationprimer or a primer pair, “stringent conditions” are conditions thatpermit the primer or primer pair to hybridize to the targetpolynucleotide to amplify the HCEM 3′ region and chromosome 10 region,or, with a primer pair, in which one primer having the correspondingwild-type sequence and another primer having the corresponding HCEM485inserted 3′ DNA sequence. Stringent conditions are sequence-dependentand will be variable in different circumstances. By controlling thestringency of the hybridization and/or washing conditions, targetsequences that are 100% complementary to the probe can be identified(homologous probing). Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofidentity are detected (heterologous probing). Generally, a probe is lessthan about 1000 nucleotides in length or less than 500 nucleotides inlength.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. One skilled in the art can use a variety ofconditions of hybridization to achieve different degrees of selectivitytoward the target sequence. See e.g., Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically,stringent conditions will be those in which the salt concentration isless than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 50° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 0.1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with 90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and Haymes et al. (1985) In: Nucleic AcidHybridization, a Practical Approach, IRL Press, Washington, D.C.

Various methods can be used to detect the HCEM485 specific region oramplicon thereof, including, but not limited to, Genetic Bit Analysis(Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167-4175) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking sequence) a single-stranded PCR product can beannealed to the immobilized oligonucleotide and serve as a template fora single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge (2000) “Pyrosequencing—a new approach to DNA analysis” Innov.Pharma. Tech. 00: 18-24. In this method, an oligonucleotide is designedthat overlaps the adjacent DNA and insert DNA junction. Theoligonucleotide is annealed to a single-stranded PCR product from theregion of interest (one primer in the inserted sequence and one in theflanking sequence) and incubated in the presence of a DNA polymerase,ATP, sulfurylase, luciferase, apyrase, adenosine 5′ phosphosulfate andluciferin. dNTPs are added individually and the incorporation results ina light signal which is measured. A light signal indicates the presenceof the insert/flanking sequence due to successful amplification,hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen et al. ((1999) GenomeRes. 9: 492-498) is also a method that can be used to detect an ampliconof the invention. Using this method, an oligonucleotide is designedwhich overlaps the flanking and inserted DNA junction. Theoligonucleotide is hybridized to a single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

TaqMan® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. (1996) Nature Biotech. 14: 303-308. Briefly,a FRET oligonucleotide probe is designed that overlaps the flanking andinsert DNA junction. The unique structure of the FRET probe results init containing secondary structure that keeps the fluorescent andquenching moieties in close proximity. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking sequence)are cycled in the presence of a thermostable polymerase and dNTPs.Following successful PCR amplification, hybridization of the FRET probeto the target sequence results in the removal of the probe secondarystructure and spatial separation of the fluorescent and quenchingmoieties. A fluorescent signal results. A fluorescent signal indicatesthe presence of the flanking/insert sequence due to successfulamplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

As used herein, “kit” refers to a set of reagents for the purpose ofperforming the method embodiments of the invention, more particularly,the identification and/or the detection of the HCEM485 event inbiological samples. The kit of the invention can be used, and itscomponents can be specifically adjusted, for purposes of quality control(e.g. purity of seed lots), detection of event HCEM485 in plantmaterial, or material comprising or derived from plant material, such asbut not limited to food or feed products.

In specific embodiments, a kit for identifying event HCEM485 in abiological sample is provided. The kit comprises a primer or a primerpair of a first and a second primer, wherein the primer or first andsecond primer amplify a polynucleotide comprising a HCEM485 specificregion. In further embodiments, the kit also comprises a polynucleotidefor the detection of the HCEM485 specific region. The kit can comprise,for example, a primer comprising SEQ ID NO: 5 (which functions as bothforward and reverse primer and can be used alone, in a preferredembodiment, under low stringency conditions); or a primer pair, thefirst primer comprising a sequence of or a fragment of a polynucleotideof SEQ ID NO: 2, 4 or 7, wherein the first or the second primer sharessufficient sequence homology or complementarity to the polynucleotide toamplify said HCEM485 specific region. For example, in specificembodiments, the first primer comprises a fragment of a polynucleotideof SEQ ID NO: 2, 7, 8, or 9, wherein the first or the second primershares sufficient sequence homology or complementarity to thepolynucleotide to amplify the HCEM485 specific region. The fragment cancomprise 10, 20, 30, 40, 50, 60, 70, or greater consecutive nucleotides.In still further embodiments, the first and the second primer cancomprise any one or any combination of the sequences set forth in SEQ IDNO: 5, 6, 10, 11, 18 or 19. The primers can be of any length sufficientto amplify the HCEM485 region including, for example, at least 6, 7, 8,9, 10, 15, 20, 15, or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30,30-35, 35-40, 40-45 nucleotides or longer. In other embodiments, SEQ IDNO: 7 or a fragment thereof or any region of SEQ ID NO: 2 can be used asa probe. Such fragments can be used as a probe having at least 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 orgreater consecutive nucleotides of SEQ ID NO:7.

Further provided are DNA detection kits. A detection kit can, forexample, include probes and/or primers directed to and/or which comprisejunction sequences. Such primers are typically at least about 10 or 15nucleotides ore more in length. An embodiment provides for nucleotidesequence comprising at least about 10 or 15 nucleotides of a portion ofthe insert, or complements thereof, and a similar length of the flankinggenomic DNA or complements thereof. As noted, the invention includes aprimer which is capable of providing amplification of a sequence whichidentifies the presence of the HCEM event, such as Primer 302 (SEQ IDNO: 5). Another option is to have a primer pair, where one primerhybridizes in the flanking region and one primer hybridizes in theinsert. One skilled in the art appreciates that the primer or probe maynot be perfectly complementary to the sequence yet be readily employedin the invention. A degree of mismatch may be tolerated as long as theyare diagnostic for the event. By way of example, without limitation,with a 20 nucleotide primer, hybridization may yet occur when one or twonucleotides do not bind with the opposite strand, if the base isinternal or on the end of the primer opposite the amplicon. In anembodiment, the kit comprises at least one polynucleotide that canspecifically detect a HCEM485 specific region or insert DNA, whereinsaid polynucleotide comprises at least one DNA molecule of a sufficientlength of contiguous nucleotides homologous or complementary to SEQ IDNO: 2, 4 or 7, and in another comprises at least one polynucleotide thatcan specifically detect a maize chromosome 10 region, wherein saidpolynucleotide comprises at least one DNA molecule of a sufficientlength of contiguous nucleotides homologous or complementary to SEQ IDNO: 8 or 9. In specific embodiments, the DNA detection kit comprises apolynucleotide having SEQ ID NO: 4, 12, 13, 14, 15, 16, or 17 and/orcomprises a sequence which hybridizes with sequences selected from thegroup consisting of SEQ ID NO: 8 or 9. In an embodiment of theinvention, a kit can comprise the 302 primer (SEQ ID NO: 5) and maycomprise a primer pair selected from the group consisting of SEQ ID NO:5, 6, 10, 11, 18 and 19. In an embodiment the kit may comprise SEQ IDNO: 5 and 10. In another embodiment the kit may comprise SEQ ID NO: 18and 19 and may include SEQ ID NO: 20.

Zygosity of a plant comprising the event can also be determined usingthe primers described here. Two primers recognizing the wild-type locusbefore integration are designed in such a way that they are directedtowards each other and have the insertion site located in between theprimers. These primers may be primers specifically recognizing flankingsequences. Together with a primer complementary to transforming DNAallow simultaneous diagnostic PCR amplification of the HCEM485 specificlocus, as well as of the wild type locus. If the plant is homozygous forthe transgenic/cisgenic locus or the corresponding wild type locus, thediagnostic PCR will give rise to a single PCR product typical,preferably typical in length, for either the transgenic or wild typelocus. If the plant is hemizygous for the transgenic locus, twolocus-specific PCR products will appear, reflecting both theamplification of the transgenic/cisgenic and wild type locus.

Plants comprising the HCEM event have as a characteristic tolerance toapplication of glyphosate (N-phosphonomethylglycine). When referring toglyphosate, the term should be considered to include any herbicidallyeffective form of N-phosphonomethylglycine and any salt thereof andforms which result in the production of the glyphosate zwitterion inplanta. Glyphosate is a competitive inhibitors of5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) or EPSPS withrespect to the binding of PEP (phosphoenolpyruvate). After theapplication of phosponomethylglycine herbicide to the plant, it istranslocated in the plant where it accumulates in the rapidly growingparts, in particular the cauline and root apices, causing damage to thepoint of destruction of sensitive plants. Depending upon the applicationrate of the herbicide, the sensitive plant growth is inhibited, that is,its growth is slowed or stopped completely. When referring to resistanceor tolerance to the glyphosate herbicide, it is meant that any impact ofthe herbicide on the plant does not kill the plant; there can be minimalimpact on the plant or no impact at all, such that any adverse impact onthe plant comprising the inserted nucleic acid molecule providingresistance or tolerance is less than in a plant not comprising a nucleicacid molecule providing resistance or tolerance to glyphosate.

The present invention provides methods for controlling weeds in an areaof cultivation, preventing the development or the appearance ofherbicide resistant weeds in an area of cultivation, producing a crop,and increasing crop safety. The term “controlling,” and derivationsthereof, for example, as in “controlling weeds” refers to one or more ofinhibiting the growth, germination, reproduction, and/or proliferationof, and/or killing, removing, destroying, or otherwise diminishing theoccurrence and/or activity of a weed. As used herein, an “area ofcultivation” comprises any region in which one desires to grow a plant.Such areas of cultivations include, but are not limited to, a field inwhich a plant is cultivated (such as a crop field, a sod field, a treefield, a managed forest, a field for culturing fruits and vegetables,etc), a greenhouse, a growth chamber, etc.

The methods of the invention comprise planting the area of cultivationwith the maize HCEM485 seeds or plants, and in specific embodiments,applying to the crop, seed, weed or area of cultivation thereof aneffective amount of a glyphosate composition and, where applicable,another herbicide or chemical of interest either at the same time or atseparate times. It is recognized that the herbicide can be appliedbefore or after the crop is planted in the area of cultivation. Suchherbicide applications can include an application of glyphosate, anyother applicable chemical, or any combination thereof.

In another embodiment, the method of controlling weeds comprisesplanting the area with a HCEM485 maize crop seed or plant and applyingto the crop, crop part, seed of said crop or the area under cultivation,an effective amount of a herbicide, wherein said effective amountcomprises a level that is above the recommended label use rate for thecrop, wherein said effective amount is tolerated when applied to theHCEM485 maize crop, crop part, seed, or the area of cultivation thereof.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell, and may be any suitable plant or plant cell. A controlplant or plant cell may comprise, for example: (a) a wild-type plant orcell, i.e., of the same genotype as the starting material for thegenetic alteration which resulted in the subject plant or cell; (b) aplant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell which is genetically identical to thesubject plant or plant cell but which is not exposed to the sametreatment (e.g., herbicide treatment) as the subject plant or plantcell; (e) the subject plant or plant cell itself, under conditions inwhich the gene of interest is not expressed; or (f) the subject plant orplant cell itself, under conditions in which it has not been exposed toa particular treatment such as, for example, a herbicide or combinationof herbicides and/or other chemicals. In some instances, an appropriatecontrol plant or control plant cell may have a different genotype fromthe subject plant or plant cell but may share the herbicide-sensitivecharacteristics of the starting material for the genetic alteration(s)which resulted in the subject plant or cell (see, e.g., Green (1998)Weed Technology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516). In other embodiments, the null segregant can be used as acontrol, as they are genetically identical to HCEM485 with the exceptionof the insert DNA.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. “Weed” as used herein refers to a plant which is notdesirable in a particular area. Conversely, a “crop plant” as usedherein refers to a plant which is desired in a particular area, such as,for example, a maize plant. Thus, in some embodiments, a weed is anon-crop plant or a non-crop species, while in some embodiments, a weedis a crop species which is sought to be eliminated from a particulararea, such as, for example, an inferior and/or non-genetically modifiedmaize plant in a field planted with maize event HCEM485, or a maizeplant in a field planted with HCEM485.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cropplants are significantly damaged or killed.

In specific embodiments, a glyphosate composition is applied to themaize HCEM485, wherein the effective concentration of the glyphosatecomposition would significantly damage an appropriate control plant.

As disclosed elsewhere herein, any effective amount of these herbicidescan be applied and is readily known by one skilled in the art. In someembodiments of the invention, glyphosate is applied to an area ofcultivation and/or to at least one plant in an area of cultivation atrates between 8 and 32 ounces of acid equivalent per acre, or at ratesbetween 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 ounces of acidequivalent per acre at the lower end of the range of application andbetween 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 ounces of acidequivalent per acre at the higher end of the range of application (1ounce=29.57 ml). In other embodiments, glyphosate is applied at least at1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater ounce of activeingredient per hectare (1 ounce=29.57 ml). Additional ranges of theeffective amounts of herbicides can be found, for example, in variouspublications from University Extension services. See, for example,Bernards et al. (2006) Guide for Weed Management in Nebraska(www.ianrpubs.url.edu/sendlt/ec130); Regher et al. (2005) Chemical WeedControl for Fields Crops, Pastures, Rangeland, and Noncropland, KansasState University Agricultural Extension Station and Corporate ExtensionService; Zollinger et al. (2006) North Dakota Weed Control Guide, NorthDakota Extension Service, and the Iowa State University Extension atwww.weeds.iastate.edu.

The herbicide is applied in any manner appropriate for thecircumstances, whether prior to the plant emerging, or after the plantemerges. In one embodiment, methods are provided for coating seeds. Themethods comprise coating a seed with an effective amount of an herbicideor a combination of herbicides (as disclosed elsewhere herein). Themethods of the invention encompass the use of simultaneous and/orsequential applications of multiple classes of herbicides. In someembodiments, the methods of the invention involve treating a plant ofthe invention and/or an area of interest (e.g., a field or area ofcultivation) and/or weed with just one herbicide or other chemical suchas, for example, a sulfonylurea herbicide. The composition applied tothe plants may include any other desirable ingredients, such asadditional chemicals such as insecticides or other herbicides or thelike, adjuvants, surfactants, or other desired component. Further,compositions may be provided at the same time or sequentially toapplication of the glyphosate herbicide, as noted, which can be anycomposition that is agronomically desirable, such as fertilizer, asecond herbicide, an insecticide, or the like.

The following is provided by way of illustration and is not intended tobe limiting to the scope of the invention.

Example 1

Glyphosate herbicide-tolerant maize line HCEM485 was produced byintroducing a 6.0 kb maize genomic fragment containing a modified formof the endogenous maize EPSPS encoding gene. The sequence providingtolerance to glyphosate exposure is described at U.S. Pat. No.7,045,684, incorporated herein by reference in its' entirety. Thesequence is SEQ ID NO: 2. The 6.0 kb fragment includes an EPSPS 5′regulatory sequence (the sequence before nucleotide 1868), an EPSPSexons and introns sequence (from nucleotide 1868 to nucleotide 5146),and an EPSPS 3′ regulatory sequence (the sequence after nucleotide5146). The EPSPS coding sequence also encodes a putative chloroplasttransit peptide (from nucleotide 1868 to nucleotide 2041). The twomutations introduced into the corn wild-type EPSPS gene are a cytosineto thymine substitution at nucleotide 2886, and a cytosine to thyminesubstitution at nucleotide 2897. The mutated gene (referred to as HCEM)encodes a mutant protein which is SEQ ID NO: 3 with the residue atposition 164 changed from threonine in the wild-type to isoleucine (Thrto Ile) and at position 168 changed from proline to serine (Pro to Ser).The resulting mutated protein was glyphosate resistant.

The mutated nucleotide sequence of SEQ ID NO: 2 includes 2mEPSPS, thatis, in this instance, the native corn EPSPS promoter, coding region(containing the two mutations), introns and 3′ terminator region. FIG. 1shows the plasmid map of pHCEM containing the 6.0 kb ClaI-EcoRV fragmentcloned into pBlueScript vector. The positions of relevant restrictionendonuclease sites and of probes used in Southern hybridization analysesare indicated with numbering relative to the plasmid DNA sequence. ForDNA introduction, pHCEM was digested with ClaI and EcoRV, subjected toagarose gel electrophoresis (1 percent agarose), and the 6.0 kb band wasexcised and purified using Qiagen's Qiaquick gel extraction kit.

This was introduced into a corn plant as described in Example 4 of the'684 patent, incorporated herein by reference in its entirety, andresulted in event HCEM485 wherein the introduced DNA is located onchromosome 10. DNA introduction was via aerosol beam injector, which isa naked DNA delivery method. The purified maize DNA fragment wasintroduced into immature maize embryos derived from the elite inbredline Stine 963 by aerosol beam injection. Aerosol beam technologyemploys the jet expansion of an inert gas as it passes from a region ofhigher gas pressure to a region of lower gas pressure through a smallorifice. The expanding gas accelerates aerosol droplets containing themolecules to be introduced into a cell or tissue. DNA carried in aerosoldroplets of this small size penetrates cells only because of the speedsattained by the aerosol droplets. Speeds achieved by the aerosol beammethod of the invention are supersonic and can reach 2,000meters/second. In a preferred embodiment, the process includes (I)culturing a source of cells, (II) optionally, pretreating cells to yieldtissue with increased capacity for uptake and integration by aerosolbeam technology, (III) transforming said tissue with an exogenousnucleotide sequence by the aerosol beam method of the invention, (IV)optionally, identifying or selecting for transformed tissue, (V)optionally regenerating transgenic plants from the transformed cells ortissue, and (VI) optionally, producing progeny of said transgenicplants. This process is described in detail at Held et al., U.S. Pat.Nos. 6,809,232; 7,067,716; and 7,026,286, incorporated herein byreference in their entirety. After 5 days of culture on non-selectivemedium, embryos were transferred onto medium containing glyphosate (100mg/l). After two 14-day passages, embryos were transferred onto mediumcontaining successively greater glyphosate concentrations, up to 540mg/1, and regeneration was carried out as previously described (See '684patent)

Example 2

The introduced sequences in maize line HCEM485 are contained within asingle genetic locus within the maize genome as demonstrated by Southernblot analysis and Mendelian inheritance studies. The modified maize EPSPsynthase expressed in maize line HCEM485 is intact, of the expectedmolecular weight and there was no evidence of truncated forms of theenzyme.

Southern analysis of HCEM485 maize DNA was performed in order toestimate the number of sites of insertion of the introduced DNA. Twoprobes were used that together spanned the entire 6.0 kb maize DNAfragment introduced into HCEM485. These probes were designated:

a) A/C—obtained from a double digest of the pHCEM plasmid with ClaI andAcc65I (corresponding to positions 1-2346) (SEQ ID NO: 21); and

b) A/E—obtained from a double digest of the pHCEM plasmid with Acc65Iand EcoRV (corresponding to positions 2347-6010) (SEQ ID NO: 22) Probes(ca. 50 ng each) were labeled with 50 μCi of (α-32P)-dCTP (3000 Ci/mmol)using a random labeling system (Rediprime™ II, Amersham Piscataway,N.J.). Genomic DNA (7 μg) isolated from HCEM485 and control Stine 963maize was digested (37° C., overnight) with HindIII and restrictionfragments were separated by agarose gel electrophoresis followed bytransfer onto Hybond N+ nylon membrane. Southern hybridizations wereperformed according to standard procedures.

Southern analysis of HCEM485 genomic DNA using both the A/C (FIG. 2,lane 4) and A/E (FIG. 2, lane 2) probes following HindIII digestionindicated the presence of a single >=23 kb hybridizing fragment that wasunique to HCEM485 (i.e., not present in digests of control Stine 963maize DNA). As there are no HindIII sites within the 6.0 kb maize DNAfragment introduced into HCEM485 and based on results from Southernanalyses using other restriction endonucleases, it is postulated thatmultiple copies of the 6.0 kb fragment, approximately 4, were insertedat a single site within the maize genome.

Example 3

The Line 963 HCEM485 plant was selfed over two generations and crossedwith line 9289, yielding F₁ hybrids hemizygous for the insert. A singleplant from this cross was crossed with line 9032, yielding F₁populations expected to segregate 1:1 for the glyphosate tolerancetrait. The F₂ generation was produced by selfing a single trait-positiveplant from the preceding F₁ generation. Progeny F₂ plants shouldsegregate 3:1 for the glyphosate-tolerance trait.

Segregation analysis was conducted on F₁ and F₂ segregating plantpopulations derived from maize line HCEM485 as described by screeningfor glyphosate tolerance. Progeny plants of each generation were grownin the greenhouse and treated with 2.5× the recommended fieldapplication rate of glyphosate at approximately the V4 stage of plantdevelopment and visually scored for herbicide susceptibility. Numbers oftrait positive and trait negative plants from each generation are shownin Table 1.

TABLE 3 Observed vs. expected segregants for F1 hybrid and F2 selfedgenerations derived from HCEM485 maize (9289xHCEM485)9032(9289xHCEM485)9032 F1 S1F2 Observed Expected Observed Expected TraitPositive¹ 129 124.5 107 108 Trait Negative 120 124.5 37 36 Total 249 249144 144 Expected 1:1 3:1 Segregation Ratio Observed 1.036:0.9642.972:1.028 Segregation Ratio χ² 0.930 0.624 ¹Differentiation of traitpositive and trait negative plants was based on tolerance to glyphosate.Plants were sprayed at the V4 stage of development with 2.5X the normalrate of glyphosate application (1X = 32 oz/acre). ²For significance atthe 95% confidence level (p < 0.05), the Chi square value should be >=3.841. Chi square values <3.841 indicate that the null hypothesis (i.e.,observed and expected segregation ratios are not significantlydifferent) should not be rejected at the 95% confidence level.The data in Table 1 were used to assess the goodness-of-fit of theobserved ratios to the expected ratios using Chi Square analysis withYates correction factor.χ²=Σ[Observed-expected−0.5]2/expectedThis analysis tested the hypothesis that the introduced trait segregatedas a single locus in a Mendelian fashion. The critical value to rejectthe hypothesis at the 5% level is 3.84. Since the Chi squared value wasless than 3.84 (Table 1), the hypothesis that the genetic trait behavedin a Mendelian fashion was accepted.

Example 4

In order to confirm the absence of any plasmid backbone sequences withinthe HCEM485 genome, samples of genomic DNA from plants of the T₂generation described in Example 3. The samples were digested withHindIII followed by Southern hybridization using the C/E probe, whichwas complementary to the plasmid backbone sequences in vector pHCEM.There were no detectable hybridization signals from samples derived frommaize line HCEM485, consistent with the lack of incorporation of anyvector backbone derived sequences in the maize genome.

Example 5

A Western blot analysis was conducted with a monoclonal antibodyspecific to 2mEPSPS to assess the integrity of the expressed protein inmaize line HCEM485. The Western blot analysis demonstrated that the2mEPSPS protein expressed in leaf and seed tissues from line HCEM485 wasintact and was of the expected size corresponding to the EPSPS protein.There were no cross-reacting species detected in control samples ofparental Stine 963 maize, indicating that the monoclonal antibody usedfor detection was specific for the modified form of the maize EPSPsynthase. The monoclonal antibody used is that described at U.S. Pat.No. 7,807,791 which is incorporated herein by reference in its entirety.The antibody is useful in identifying the presence of the 2mEPSPS gene.

Example 6

Agronomic and phenotypic characteristics of an HCEM485 maize hybrid andthree control hybrids were evaluated in a series of field trials across15 United States Corn Belt locations. These trials used the followingcomparisons:

-   -   HCEM485 hybrid (((HCEM485)2/9289/9032)3/5056) [trait positive]        (see FIG. 3)        -   Control hybrid 9289×5056 [trait negative]        -   Control hybrid 9032×5056 [trait negative]        -   Control hybrid 963×5056 [trait negative]

Up to 17 separate agronomic characteristics were assessed at eachlocation, but not all traits were assessed at all locations. Theseagronomic traits covered a broad range of characteristics encompassingthe entire life cycle of the maize plant and included data assessinggermination and seedling emergence, growth habit, vegetative vigor, daysto pollen shed, days to maturity, and yield parameters

Parameters used to evaluate yield and grain characteristics included:YGSMN (grain yield); HAVPN (plant population at harvest); DROPP (percentdropped ears); TWSMN (grain test weight); and GMSTP (grain moisturepercent). Among the varieties suitable for statistical analysis, therewere no significant differences in average yield, plant population atharvest, grain moisture, or grain test weight between HCEM485 andcontrol hybrids (Table 8). For both yield and plant population atharvest, there were significant genotype x location interactions.Although not subject to statistical analysis, there were no remarkabledifferences in percent dropped ears between HCEM485 and controlgenotypes (Table 2).

TABLE 2 Comparison of yield and grain characteristics of HCEM485 andcontrol hybrids YGSMN^(a) HAVPN DROPP GMSTP TWSMN (bu/acre)(plants/acre) (%) (%) (lb/bu) HCEM485 hybrid 115.4 ± 51.2 14976 ± 34100.06 18.8 ± 7.7 55.2 ± 1.9 Control hybrids 113.9 ± 50.7 14888 ± 36220.04 18.3 ± 7.1 55.2 ± 2.3 Mean Difference 1.5 88 0.02 0.5 0.1 F-testgenotype 0.621 0.818 0.051 0.731 F-test genotype × location 0.003**0.040** 0.463 0.431 N^(b) 13 14 14 13 13 **= indicates that the effectof genotype was not consistent across all locations, in which case thecomparison of genotype averaged across locations is questionable.^(a)YGSMN = grain yield; HAVPN = final stand count at harvest; DROPP =percent dropped ears; GMSTP = grain moisture percent; TWSMN = grain testweight. Mean values are shown. For YGSMN, HAVPN and GMSTP, the meanstandard deviation is indicated. ^(b)N = number of locations with data.

In summary, the agronomic characteristics chosen for comparison werethose typically observed by professional maize breeders and agronomistsand represented a broad range of characteristics throughout thedevelopment of the maize plant. Results of these trials suggest thatthere were no biologically significant unintended effects on plantgrowth habit and general morphology, vegetative vigor, flowering andpollination, grain yield, grain test weight, or disease susceptibilityas a result of the genetic modification introduced into maize lineHCEM485.

Example 7

Flanking sequence of the integrated DNA fragment called HCEM in eventHCEM485 was sought for the purpose of generating event specific PCRbased assays. The HCEM DNA fragment contains a double mutant epsps gene(2mepsps) including the promoter region, introns, exons, and 3′terminator region (U.S. Pat. No. 7,045,684). HCEM485 DNA and control DNAfrom Inbred 963 was obtained from leaves of corn plants using ‘PlantDNeasy Kit’ from Qiagen. Ample concentrated DNA was obtained in a highlypure form from both the controls and the HCEM485 plants using the Qiagenprotocol. Primers were designed for amplification of DNA using segmentsof known sequence from the HCEM DNA fragment. Primers designed for the3′ end of HCEM were named in the 300's. The complete sequence of therelevant primer (302) is listed below:

(SEQ ID NO: 5) Primer 302: ATGTTACTATGGTGCCTTCTTATCC

In addition, a random 9-mer was used in an attempt to anchor thereaction in the unknown flanking sequence. A PCR process was used toamplify the desired segment of DNA, which produced a clear band when theHCEM485-exhibitor DNA sample was run on a gel. No corresponding bandresulted from the control DNA under identical conditions. The PCRprocess was TAIL PCR (Thermal Asymmetric Interlaced Polymerase ChainReaction), and used two separate steps to isolate flanking sequence. ThePCR proceeded as follows:

First Stage PCR:

25 ul Qiagen Taq PCR Master Mix

23.5 ul H₂O

1 ul Primer 302 at a concentration of 1 ug/ul H₂O

1 ul DNA (485) at a concentration of 1 ug/ul H₂O

First Stage Cycle Parameters:

Cycle 1:

2 minutes at 95° Celsius

1 Repetition

Cycle 2:

25 seconds at 95° Celsius

35 seconds at 60° Celsius

2 minutes and 30 seconds at 70° Celsius

35 repetitions

Second Stage PCR:

25 ul Qiagen Taq PCR Master Mix

20 ul H₂O

10 ul 485 (302) PCR product

1 ul random 9-mer at a concentration of 1 ug/ul H₂O

1 ul primer 302 at a concentration of 1 ug/ul H₂O

Second Stage Cycle Parameters:

Cycle 1:

2 minutes at 95° Celsius

1 Repetition

Cycle 2:

25 seconds at 95° Celsius

35 seconds at 45° Celsius

1 minute 45 seconds at 70° Celsius

35 Repetitions

The resultant reaction mixtures were run on a gel with a DNA marker anda band of about 1,100 base pairs was identified for the HCEM485 sample.No band of that size was identified for the 963 control. The DNA bandwas extracted from the gel using “Gel Extraction Kit” protocol fromQiagen. The DNA was cloned into “pGEM T-Easy” plasmid (Promega)according to manufacturer instructions. The resulting plasmid wasisolated using a Qiagen “Maxi-Prep Procedure” and called J4. The samplewas sent to the ISU DNA Facility for nucleotide sequence analysis. Thesequence illustrated in FIG. 4 was obtained. The J4 fragment is 1112base pairs in length and is flanked by primer 302 which indicates thereaction was anchored on both ends by primer 302. Primer 302 (SEQ ID NO:5) is shown underlined at the beginning of the sequence. The regioncomplementary to Primer 302 is underlined at the end of the sequence inFIG. 4 (SEQ ID NO: 6). The first 367 nucleotides have 100% homology tothe 3′ sequence of the HCEM fragment (SEQ ID NO: 7). The junction atpositions 367/368 is italicized and bolded in FIG. 1. The rest of the J4sequence (positions 368-1112, SEQ ID NO: 8) was blasted against theentire corn genome (www.maizesequence.org/blast). This revealed 100%homology over the length of 725 continuous base pairs (positions368-1092, SEQ ID NO: 9) to a section of the tenth chromosome, allowingus to conclude that the HCEM fragment was inserted in the tenthchromosome. Downstream of position 1092 (positions 1093-1112) of SEQ IDNO: 4 the homology is much less to the corn genome continuous to theregion of 100% homology (non-homology is displayed by small letters inFIG. 1). However, the homology was apparently enough to allow primingwith the 302 primer at the low annealing temperature of 45° C. FIG. 5 isa graphic representation of location of the regions in the plasmid.

Primers were designed using the identified flanking region. The sequenceof the relevant primer (506) is listed below and its compliment appearsin bold font in Seq. 1:

(SEQ ID NO: 10) Primer 506: CGCCCAGTAGGTACACTAAG

This primer was used in combination with primer 302 in a PCR procedureto verify difference between the control DNA Inbred 963 and the HCEM485exhibitor. The reaction was set up as follows:

PCR:

12.5 ul Qiagen Taq PCR Master Mix

11 ul H₂O

0.5 ul Primer 302 at a concentration of 1 ug/ul H₂O

0.5 ul Primer 506 at a concentration of 1 ug/ul H₂O

1 ul DNA (HCEM485 or Inbred 963) at a concentration of 1 ug/ul H₂O

Cycle Parameters:

Cycle 1:

2 minutes at 90° Celsius

1 Repetition

Cycle 2:

25 seconds at 90° Celsius

35 seconds at 45° Celsius

2 minutes 30 seconds at 72° Celsius

35 Repetitions

This reaction yielded a band of about 1,000 base pairs when performedwith template DNA from HCEM485, while no such band resulted from thereaction containing DNA from Inbred 963. Due to the expected size andthe fact that it is unique to HCEM485, we conclude that the reaction isevent-specific.

Example 8

The presence of the HCEM485 Maize transformation event in genomic DNAextracted from seed tissue was confirmed using a TaqMan® assay. Specificdetection of the HCEM485 event used PCR amplification of the region thatspans a junction site of the HCEM485 insert and genomic flankingsequences (see FIGS. 4 and 5). Amplification was achieved using twospecific primers which amplify a 121 bp DNA fragment (Table 5).Amplification was measured with a target-specific MGB probe containingthe FAM reporter. The protocol below outlines the reaction reagents, theoligonucleotide primers and probes, and the thermocycling conditionsused to perform the reaction.

Preparation of DNA Template

DNA was extracted from ground maize seed using the Plant DNeasy Kit fromQiagen. DNA was quantified and diluted to a final concentration of 25ng/ul with DNase-free water. It is recommended to use control DNA thatwas extracted and normalized using the same method as the samples to beanalyzed. The controls for this analysis included positive control fromHCEM485 transgenic maize, negative control from non-transgenic maize andnegative control that contains no template DNA.

Polymerase Chain Reaction

A. Prepare Reaction Mixture

The procedure involved determining the number of reactions to beperformed, including controls and prepare a master mix consisting of allcomponents of the reaction, except the template, to supply all reactionsplus 10% excess.

TABLE 3 Volume Final per Final PCR reagent Conc. reaction Volume ddH2O.31 TaqMan Gene Exp PCR MM (2X) 1X 10 HCEM485 Forward Primer SB060 (250.9 uM 0.72 uM) HCEM485 Reverse Primer SB061 (25 0.9 uM 0.72 uM) HCEM485Probe SBTM021 (20 uM) 0.25 uM  0.25 Total Volume 1212 ul of PCR reaction mix was aliquoted to each PCR tube/well. 8 ul ofDNA (25 ng/ul) of each sample and control was added to individual PCRtube/well and mixed well by pipetting up and down. The reactiontube/plate was sealed and centrifuged at low speed to spin down thereaction mixture.

B. PCR Amplification

The following cycling parameters were used with the Applied BiosystemsStep-One Plus Real-Time PCR System.

TABLE 4 Cycle Data Temperature Time No. Collection 60° C. 30 Sec 1 Yes50° C.  2 Min 1 No 95° C. 10 Min 1 No 95° C. 15 Sec 40 Yes 60° C.  1 Min60° C. 30 Sec 1 Yes

TABLE 5 PCR primers and probes Name Description Sequence 5′ to 3′ SB060Forward Primer targeted to CATTGAAAGGCATCTTAGCAATGTCTAAAthe T-DNA sequence (SEQ ID NO: 18) SB061 Reverse Primer targeted toCCACCCAGTCTCACTCAATCTAATACTATAT the genomic flanking (SEQ ID NO: 19)sequence SBTM021 6-FAM MGB probe targetedCCAAGCCCTATAAGACATCAA (SEQ ID NO: to the T-DNA/ flanking 20)sequence junctionPCR reagents2×TaqMan® Gene Expression Master Mix (Applied Biosystems)PCR primers, HPLC purified (Integrated DNA Technologies)PCR probe (Applied Bio systems)EquipmentThermocycler: Applied Biosystems Step-One Plus Real-Time PCR System

The PCR assay was optimized and validated for use in 96-well formatusing an Applied Biosystems Step-One Plus Real-Time PCR System. Othersystems may be used, but thermal cycling conditions must be verified.Event specificity of the assay was tested against DNA extracted fromeight different genetic backgrounds, one of which was a HCEM485 line.The HCEM485 line resulted in an amplification signal while no signal wasdetected in any negative control sample within 50 amplification cycles.

Sensitivity of the assay was tested against DNA extracted from pooledseed samples with varying amounts of HCEM485 presence. Seed samplesconsisted of HCEM485 seed pooled with non-transgenic conventional maizeseed at ratios of 10:0, 1:10, 1:25, 1:50, and 1:100(HCEM485:non-transgenic seed). The assay was shown to reproduciblydetect HCEM485 in all seed combinations tested. Performance was verifiedusing positive and negative blind samples. HCEM485 was detected in allpositive controls and blind samples containing HCEM485 while negativecontrols and negative blind samples did not produce a signal.

LIST OF SEQUENCES

SEQ ID NO: 1 genomic EPSPS wild-type corn fragment

SEQ ID NO: 2 mutated corn EPSPS nucleotide sequence HCEM

SEQ ID NO: 3 amino acid sequence encoded by SEQ ID NO: 2

SEQ ID NO: 4 amplified nucleotide sequence of the flanking region of the3′ sequence of HCEM and flanking region of chromosome 10.

SEQ ID NO: 5 302 primer

SEQ ID NO: 6 sequence complement to 302 primer

SEQ ID NO: 7 first 367 bp of SEQ ID NO: 4

SEQ ID NO: 8 bp 368-1112 of sequence 4

SEQ ID NO: 9 bp 368-1092 of sequence 4

SEQ ID NO: 10 506 primer

SEQ ID NO: 11 sequence complement to 506 primer

SEQ ID NO; 12 junction region—last 10 bp of HCEM flanking region andfirst 10 of chromosome 10 flanking region

SEQ ID NO: 13 junction region—last 20 bp of HCEM flanking region andfirst 20 of chromosome 10 flanking region

SEQ ID NO: 14 junction region—last 30 bp of HCEM flanking region andfirst 30 bp of chromosome 10 flanking region.

SEQ ID NO: 15 junction region—entire HCEM flanking region, bases 1-367of SEQ ID NO: 4, and chromosome 10 flanking region by 368-1092 ofsequence 4

SEQ ID NO: 16 junction region—all of HCEM (SEQ ID NO: 2) and all ofchromosome 10 flanking region by 368-1112 (SEQ ID NO: 8)

SEQ ID NO: 17 junction region—HCEM (SEQ ID NO: 2) and bp368-1092 ofchromosome 10 flanking sequence (SEQ ID NO: 9)

SEQ ID NO: 18 Primer SB060

SEQ ID NO: 19 Primer SB061

SEQ ID NO: 20 TaqMan® 6-FAM MGB probe SBTM021

SEQ ID NO 21 Probe A/C

SEQ ID NO: 22 Probe A/E

What is claimed is:
 1. A method of identifying event HCEM485 in abiological sample, said method comprising detecting a DNA moleculecomprising at least one HCEM485 junction sequence in a biological samplewith a specific probe or at least one specific primer which binds oramplifies a DNA molecule comprising said at least one HCEM485 junctionsequence, reference seed comprising said HCEM485 event having beendeposited under ATCC accession number PTA-12014.
 2. The method of claim1, wherein said DNA molecule detected comprises a junction sequence thatis within SEQ ID NO: 4 or the full complement of SEQ ID NO:
 4. 3. Themethod of claim 1, wherein said DNA molecule comprises a junctionsequence selected from the group consisting of 4, 12, 13, 14, 15, 16 and17.
 4. The method of claim 1, wherein said DNA molecule is detected byamplification of a nucleic acid molecule using a primer selected fromthe group consisting of SEQ ID NO: 5, 6, 10, 11, 18 and 19 or the fulllength complement thereof.
 5. The method of claim 1, wherein said DNAmolecule is detected by amplification using a primer pair comprising SEQID NO: 5 and 10 or SEQ ID NO: 18 and
 19. 6. The method of claim 1,wherein said DNA molecule is detected by a probe selected from the groupconsisting of SEQ ID NO: 21 and
 22. 7. A kit for identifying thepresence of event HCEM485 in a biological sample, said kit comprising aspecific probe that binds at least the HCEM485 junction sequences or atleast one specific primer that comprises at least the HCEM485 junctionsequences, reference seed comprising said event having been depositedunder ATCC accession number PTA-12014.
 8. The kit of claim 7, whereinsaid junction sequence comprises a polynucleotide selected from thegroup consisting of SEQ ID NO: 4, 12, 13, 14, 15, 16 and
 17. 9. The kitof claim 7, wherein said probe is selected from the group consisting ofSEQ ID NO: 21 and
 22. 10. An isolated polynucleotide comprising SEQ IDNO: 4, 12, 13, 14, 15, 16 and 17.